About 1011 metric tons of chitin are produced annually in the ocean waters, 109 tons from copepods alone, the most abundant animals on earth. Chitin fibrils embedded in a matrix of highly cross-linked, insoluble protein(s) make up a significant fraction of the cuticles of arthropoda, annalida, mollusca, and the cell walls of many fungi and yeasts.
Each chitin fibril molecule consists of a straight chain of over a thousand N-acetylglucosamine (GlcNAc) monomers linked by β,1-4 bonds similar to beads on a string. These fibril molecules are cross-linked via hydrogen bonds to each other, giving enormous strength to the fibers. Native chitin also contains a small percentage of glucosamine (GlcN). Harsh chemical methods (acid and alkali) are used to isolate chitin, thus yielding an increased quantity of glucosamine in the polymer.
The ecological significance of chitin degradation has been recognized for over a century. The carbon and nitrogen cycles would cease if this highly insoluble polysaccharide was not recycled in a biologically useful form. The commercial significance of chitin breakdown has also been recognized in the context of production of bioavailable sugars and in medicine for the treatment of arthritis, and other joint problems.
Although a constant rain of chitin falls to the ocean floors (marine snow), marine sediments contain only traces of the polymer. Zobell and Rittenberg (J. Bacteriol. 35: 275-287 (1937)) demonstrated the existence of chitinivorous bacteria that cause degradation of chitin and are ubiquitous in the marine environment.
Historically, it was thought that only two steps were required to convert chitin to GlcNAc. The first step relied on a chitinase (EC:3.2.1.14) that would yield primarily the disaccharide, (GlcNAc-β,1-4-GlcNAc, or (GlcNAc)2 or N,N′-diacetylchitobiose). The second step was thought to utilize a β-N-acetylglucosaminidase (EC: 3.2.1.52) to hydrolyze the disaccharide to GlcNAc. Further investigation revealed that the chitin catabolic cascade was much more complex involving a minimum of three signaling pathways that rely on many gene products. For instance, using DNA microarray analyses (Meibom et al. Proc. Natl. Acad. Sci. U.S.A 101: 2524-2529 (2004)), some 200 genes have been shown to be involved in the growth of Vibrio cholerae on living copepods.
Traditionally, chitin is purified from invertebrate cuticles by a process that includes using dilute acid to remove the calcium from the cuticles, followed by repetitive hot strong alkali treatment, and acid extraction to remove the highly insoluble protein matrix that surrounds the chitin in the cuticles. Chitin can then be completely hydrolyzed to GlcN by refluxing it with 4N-6N HCl for 16 hours or more. The GlcN, HCl salt must then be purified, which is accomplished by fractional crystallization or ion-exchange chromatography.
Thus, the chemical isolation of GlcN as an HCl or sulfate salt is expensive and is a high energy-consuming chemical procedure presenting ecological problems associated with the disposal of large volumes of waste products. A new method of fermentation that could convert chitin into GlcNAc or GlcN on an industrial scale would facilitate the preparation of medicines and would also provide an alternative bioavailable source of sugars. Additionally, it would be valuable if these sugars could be converted to ethanol.